1. Field of the Invention
The association peptides and methods of the invention relate to the fields of molecular biology, polymer chemistry, biotechnology, and pharmacology.
2. Description of Related Art
Bodenmuller et al., 1983, EMBO 5 (8): 1825-1829, incorporated herein by reference, shows that the neuropeptide head activator (HA) dimerizes to yield a biologically inactive form of the peptide at concentrations as low as 10.sup.-13 M, indicating extremely high binding affinity. The authors observed that a fragment containing the last six amino acids of this peptide's carboxy terminus (SKVILF) (SEQ.ID NO:1) resulted in dimers that were even "more stable" than the HA itself. The article also reports the construction of HA analogs that lack the SKVILF (SEQ.ID NO:1) motif and do not dimerize and that alteration of the phenylalanine residue at the carboxy-terminus, as well as alteration of residues attached to the amino-terminus, of SKVILF (SEQ.ID NO;1) in the Ha results in the loss of dimerization activity. While peptides capable of dimerization could be quite valuable in many molecular biology methods, these results suggested that the HA peptide could not be attached to a second molecule for purposes of dimerizing the second molecule.
One important class of molecular biology methods where dimerization peptides would be of value is the class of methods for generating and screening molecular diversity. The ability to synthesize DNA chemically has made possible the construction of extremely large collections of nucleic acids, peptides, proteins, and other polymers. These collections can be screened to isolate ligands that bind biological receptors or to identify catalysts that mediate a desired reaction by binding to a substrate. In recent years, several methods have been developed for generating such libraries of compounds for screening. These methods can be grouped for discussion into methods that utilize living organisms and methods that rely on in vitro chemical synthesis.
One important method in the former category involves the display of a biological molecule, such as a peptide, antibody, or other protein (collectively referred to as "(poly)peptide") on the surface of a phage or cell. These methods typically involve establishing a physical or logical connection between each (poly)peptide and the nucleic acid that encodes the (poly)peptide; perhaps the best known method in this category involves the presentation of a (poly)peptide on the surface of a filamentous phage. The phage can be incubated with an immobilized receptor of interest, so that phage that present a (poly)peptide that binds to the receptor can be separated from phage that do not. After several rounds of affinity enrichment and phage replication, followed by isolation of the phage that bind and sequence determination of the phage nucleic acid, this method allows one to identify the sequence of (poly)peptide ligands for the receptor. Such methods are described in more detail in PCT patent publication Nos. 91/17271; 91/18980, and 91/19818, each of which is incorporated herein by reference.
Another important recombinant method for the display of (poly)peptide ligands involves the production of a fusion protein composed of a protein that specifically binds to DNA and the potential (poly)peptide ligand. In one embodiment of this method, the library of (poly)peptides is produced by transforming recombinant host cells with a vector that encodes a lac repressor/(poly)peptide fusion protein and contains a lac operator sequence. When the transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the vector that encodes the fusion protein. Upon lysis of the host cells, the fusion protein/vector complexes can be screened against a receptor in much the same way the phage are screened in the phage-based display method. See U.S. patent application Ser. No. 963,321, filed Oct. 15, 1992, which is a continuation-in-part of Ser. No. 778,233, filed Oct. 16, 1991, and Ser. No. 08/038,726, filed Mar. 25, 1993, inventor W. P. C. Stemmer, each of which is incorporated herein by reference.
In contrast to the recombinant methods, in vitro chemical synthesis provides a method for generating libraries of compounds, without the use of living organisms, that can be screened for ability to bind to a receptor. Although in vitro methods have been used for quite some time in the pharmaceutical industry to identify potential drugs, recently developed methods have focused on rapidly and efficiently generating and screening large numbers of compounds. One early method involves the synthesis of peptides on a set of pins or rods. See PCT patent publication Nos. 84/03506 and 84/03564, each of which is incorporated herein by reference. Another method involves the use of a synthesis resin or beads and a variety of flow-through containers into which the beads are placed. The containers are then exposed to monomer-coupling solutions and labeled to indicate the monomer coupling reactions to which the container has been exposed. See U.S. Pat. No. 4,631,211, incorporated herein by reference. A related method dispenses with the labeling step and separate containers for each peptide to achieve greater diversity at the cost of easy identification of a particular ligand of interest. In this method, the synthesis beads are pooled and redistributed after each set of monomer coupling reactions. After screening with a receptor, the ligands on a bead of interest must be identified by removing the ligand from the bead and determining the molecular structure of the ligand. See PCT patent publication No. 92/00091, incorporated herein by reference.
A significant improvement over this latter method involves tagging each bead with an identifier tag, such as an oligonucleotide, so as to facilitate ligand identification. This method is described in U.S. patent application Ser. No. 946,239, filed Sep. 16, 1992, which is a continuation-in-part of Ser. No. 762,522, filed Sep. 18, 1991, each of which is incorporated herein by reference. Another powerful method for generating large collections of compounds addresses the ligand identification problem by forming arrays of different compounds in a manner that places each different compound of the array at a discrete, predefined location. The location identifies each ligand. This method, called "very large scale immobilized polymer synthesis," is described in U.S. patent No. 5,143,854; PCT patent publication Nos. 90/15070 and 92/10092; U.S. patent application Ser. No. 624,120, filed Dec. 6, 1990; Fodor et al., 15 Feb. 1991, Science 251:767-773; Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-280; and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991, each of which is incorporated herein by reference.
Other systems for generating libraries of compounds have aspects of both the recombinant and in vitro chemical synthesis methods. In these hybrid methods, biological enzymes or enzyme complexes play an important role in generating the compounds, but no living organisms or cells are directly used. For example, RNA molecules with the ability to bind a particular protein (see Tuerk and Gold, 1990, Science 249:505-510; Beaudry et al., 31 Jul. 1992, Science 257:635-641; Green et al., Feb. 1991, Methods:Meth. Enz. 2 (1): 75-86; and PCT patent publication No. 91/19813, each of which is incorporated herein by reference) or a dye (see Ellington and Szostak, 1990, Nature 346:818-822, incorporated herein by reference) have been selected by alternate rounds of affinity selection and PCR amplification. A similar technique was used to determine the DNA sequences that bind a human transcription factor (see Thiesen and Bach, 1990, Nucl. Acids Res. 18:3203-3209, Beaudry and Joyce, 31 Jul. 1992, Science 257:635-641, and PCT patent publication Nos. 92/05285 and 92/14843, each of which is incorporated herein by reference). In similar fashion, the technique of in vitro translation has been used to synthesize proteins of interest (see PCT patent publication Nos. 88/08453, 90/05785, 90/07003, and 91/02076, each of which is incorporated herein by reference); this technique has also been proposed as a method to generate large libraries of peptides. See PCT patent publication Nos. 91/05058 and 92/02536, each of which is incorporated herein by reference.
There remains a need for improved methods of and reagents for constructing and screening libraries of compounds in addition to the methods described above. For instance, many monovalent receptors do not bind to relatively low (10 .mu.m) affinity ligands presented using some of the systems described above, and a means for dimerizing such receptors would be helpful. Blondel and Bedoulle, 1991, Protein Engineering 4:457-461, incorporated herein by reference, reports the design of a dimeric form of the maltose binding protein (MBP) that included a 33-residue leucine zipper motif; other methods for dimerizing proteins would be helpful, especially if the methods produced very stable dimers. Conversely, some receptors may require dimeric ligands, and a generic means for dimerizing ligands would be useful in identifying ligands that bind to such receptors. Ghadiri et al., 1992, J. Am. Chem. Soc. 114:825-831, incorporated herein by reference, reports a method for assembling a small peptide into a multimeric structure by incorporation of a 2,2'-bipyridine moiety in the peptide and the use of a metal ion to assist assembly. Simpler methods for forming multimeric peptide sequences are needed.
In addition, catalytic molecules are important in many biological processes, yet the methods above are not all ideally suited for screening to identify such compounds. Over the past several years, many scientists have proposed that catalytic biological molecules, especially catalytic antibodies, will prove invaluable in manufacturing processes for chemicals. See, e.g., PCT patent publication Nos. 90/05746, 90/05749, 90/05785, and 92/01781; U.S. Pat. No. 5,190,865; and Tawfik et al., Jan. 1993, Proc. Natl. Acad. Sci. USA 90:373-377, each of which is incorporated herein by reference. See also U.S. patent application Ser. No. 043,459, previously incorporated herein by reference. The association peptides of the present invention will prove valuable in constructing and identifying such catalytic molecules as well as in generating and screening large libraries of compounds.